pooria gill - mazandaran university of medical...
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Outline Motivation History How the AFM works
Two modes Contact Mode Non-Contact Mode Force Measurements Raster the Tip: Generating an Image Scanning Sample
OUr AFM Pictures
Examples The Good The Bad And the Ugly
Uses Topographical Analysis Thin Layer Depth RMS Roughness Calculations
Other types of Microscopy
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Other Types of SPM Techniques Lateral Force Microscopy (LFM)
Frictional forces measured by twisting or “sideways” forces on cantilever. Magnetic Force Microscopy (MFM)
Magnetic tip detects magnetic fields/measures magnetic properties of the sample.
Electrostatic Force Microscopy (EFM) Electrically charged Pt tip detects electric fields/measures dielectric and
electrostatic properties of the sample Chemical Force Microscopy (CFM)
Chemically functionalized tip can interact with molecules on the surface –giving info on bond strengths, etc.
Near Field Scanning Optical Microscopy (NSOM) Optical technique in which a very small aperture is scanned very close to
sample Probe is a quartz fiber pulled to a sharp point and coated with aluminum to
give a sub-wavelength aperture (~100 nm)
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Motivation Digitally image a topographical surface Determine the roughness of a surface sample or to
measure the thickness of a crystal growth layer Image non-conducting surfaces such as proteins and
DNA Study the dynamic behavior of living and fixed cells
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History The Scanning Tunneling Microscope (STM) was
invented by G. Binnig and H. Rohrer, for which they were awarded the Nobel Prize in 1984
A few years later, the first Atomic Force Microscope (AFM) was developed by G. Binnig, Ch. Gerber, and C. Quate at Stanford University by gluing a tiny shard of diamond onto one end of a tiny strip of gold foil
Currently AFM is the most common form of scanning probe microscopy
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Parts of AFM 1. Laser – deflected off cantilever 2. Mirror –reflects laser beam to
photodetector 3. Photodetector –dual element
photodiode that measures differences in light intensity and converts to voltage
4. Amplifier 5. Register 6. Sample 7. Probe –tip that scans sample
made of Si 8. Cantilever –moves as scanned
over sample and deflects laser beam
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How the AFM Works The AFM brings a probe in
close proximity to the surface
The force is detected by the deflection of a spring, usually a cantilever (diving board)
Forces between the probe tip and the sample are sensed to control the distance between the the tip and the sample.
van der Waals force curve
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Two ModesRepulsive (contact) At short probe-sample distances,
the forces are repulsiveAttractive Force (non-contact) At large probe-sample distances,
the forces are attractiveThe AFM cantelever can be used to
measure both attractive force mode and repulsive forces.
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3 Modes of AFMContact ModeNon-Contact ModeTapping (Intermittent
contact) Mode
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Contact Mode Measures repulsion between tip and sample Force of tip against sample remains constant Feedback regulation keeps cantilever deflection
constant Voltage required indicates height of sample Problems: excessive tracking forces applied by probe
to sample
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Non-Contact Mode Measures attractive forces between tip and sample Tip doesn’t touch sample Van der Waals forces between tip and sample
detected Problems: Can’t use with samples in fluid Used to analyze semiconductors Doesn’t degrade or interfere with sample- better for
soft samples
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Tapping (Intermittent-Contact) Mode
Tip vertically oscillates between contacting sample surface and lifting of at frequency of 50,000 to 500,000 cycles/sec.
Oscillation amplitude reduced as probe contacts surface due to loss of energy caused by tip contacting surface
Advantages: overcomes problems associated with friction, adhesion, electrostatic forces
More effective for larger scan sizes
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Force Measurement The cantilever is designed with a
very low spring constant (easy to bend) so it is very sensitive to force.
The laser is focused to reflect off the cantilever and onto the sensor
The position of the beam in the sensor measures the deflection of the cantilever and in turn the force between the tip and the sample.
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Raster the Tip: Generating an Image The tip passes back and forth in a
straight line across the sample (think old typewriter or CRT)
In the typical imaging mode, the tip-sample force is held constant by adjusting the vertical position of the tip (feedback).
A topographic image is built up by the computer by recording the vertical position as the tip is rastered across the sample.
Scan
ning
Tip
Rast
er M
otio
n
Top Image Courtesy of Nanodevices, Inc. (www.nanodevices.com)
Bottom Image Courtesy of Stefanie Roes(www.fz-borstel.de/biophysik/ de/methods/afm.html)
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Constant-force scan vs.constant-height scanConstant-force Advantages:
Large vertical range Constant force (can be
optimized to the minimum)
Disadvantages: Requires feedback
control Slow response
Constant-height Advantages:
Simple structure (no feedback control)
Fast response Disadvantages:
Limited vertical range (cantilever bending and detector dynamic range)
Varied force
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Scanning the Sample Tip brought within nanometers of
the sample (van der Waals)• Radius of tip limits the accuracy of
analysis/ resolution• Stiffer cantilevers protect against
sample damage because they deflect less in response to a small force • This means a more sensitive
detection scheme is needed• measure change in resonance
frequency and amplitude of oscillation
Image courtesy of (www.pacificnanotech.com)
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General Applications Materials Investigated: Thin and thick film coatings,
ceramics, composites, glasses, synthetic and biological membranes, metals, polymers, and semiconductors.
Used to study phenomena of: Abrasion, adhesion, cleaning, corrosion, etching, friction, lubricating, plating, and polishing.
AFM can image surface of material in atomic resolution and also measure force at the nano-Newton scale.
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What are the limitations of AFM?
AFM imaging is not ideally sharp
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Advantages and Disadvantages of AFM
Easy sample preparation Accurate height
information Works in vacuum, air, and
liquids Living systems can be
studied
Limited vertical range Limited magnification
range Data not independent of
tip Tip or sample can be
damaged
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A Better View
Now:
• Removed extreme points
• Digitally decreased the height of analysis
• Less than 1/3 as high as initial scan
•Lose resolution and data by clipping off extreme points
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Thickness of a Thin Layerof Pd on Si WaferSi/Pd step
Step (where Pd coating ends)
Systematic error
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Surface Roughness
Roughness typically measured as root mean squared (RMS)
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Carbon Nanotube Tips• Well defined shape and composition.• High aspect ratio and small radius of curvature (“perfect” tip would be a delta
function tip).• Mechanically robust.• Chemical functionalization at tip.
DNACNT Tips
Images taken from Nanodevices, Inc. (www.nanodevices.com)and Wooley, et al., Nature Biotech. 18, 760
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• STM can move atoms around on a surface.
SPM Lithography
Iron on Copper Iron on Copper
Eigler, et al. from IBM
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• Dip Pen Lithography.SPM Lithography
Mirkin, et al. from Northwestern University
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Million Cantilever Wafer
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Millipede Memory
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Cantilever Gas Sensors (Noses)
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Cantilever Gas Sensors (Noses)
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AFM Tips
80 – 320 µm
20 µm
35 µm
125 µm
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Intermolecular interactions
Schematic of the force–extension characteristics of DNA: at 65 pN the molecule is overstretched to about 1.7 times its contour length, at 150 pN the double strand is separated into two single strands, one of which remains attached between tip and surface.
MFP
MFP is specially designed for force measurementpurpose
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Adhesion Force ImagingHeight Adhesion
0102030
0 4 8 12
Hei
ght (
nm)
0.00.30.6
0 4 8 12
Ahes
ion
(V)
Albumin
Albumin
Polystyrene
Si
PS
pH 7
5 µm
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Adhesion and Hardness Imaging
PLMA/PmMl6 blend on Si imaged in waterPLMA: poly (lauryl methacrylate)PmMl6: 2-methacryloyloxyethyl phosphorylcholine-co-lauryl methacrylate (1:6)
1 µm
Height Adhesion Stiffness
Simultaneous Height, Adhesion and Stiffness maps are obtainedwith “Pulsed-Force” AFM technique.
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Conclusions How AFM works
Constant-height and constant-force scans (contact mode) Feedback control in constant-force mode Contact mode and tapping mode
Force measurements with AFM Force curves: contact part to measure hardness and adhesion to
measure intermolecular interactions Calibrations:
Detector sensitivity (nm/V) = Inverse of contact slope on a hard surface => Convert the measured T-B signal (V) to cantilever deflection (nm)
Spring constant (N/m) => Convert the cantilever deflection to force (N) [F=-kx]
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AFM : Contact Mode
Feedback Error:Deflection
Output:“Isoforce” Height
http://www.physik3.gwdg.de/~radmacher/publications/osteobla
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AFM : Tapping Mode
Feedback Error:Amplitude
Output:“Isoamplitude” Height
Evaporated gold surface
Additional Feedback:Phase
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(http://www.energosystems.ru/fgallery.htm)
SAMPLE REFERENCEgraphite Binnig, et al., Europhys. Lett. 3, 1281 (1987)
molybdenum sulfideboron nitride
Albrecht, et al., J. Vac. Sci. Tech. A 6 271 (1988)
goldsodium chloride (001)
lithium flouride
Manne, et al., Appl. Phys. Lett. 56 1758 (1990)Meyer, et al., Appl. Phys. Lett. 56 2100 (1990)
Meyer, et al., Z. Phys.B. 79 3 (1990)(1014) cleavage plane of a
calcite (CaCO3) crystalOhnesorge, et al., Science 260 1451 (1993)
Highly Oriented Pyrolytic Graphite (HOPG)
http://stm2.nrl.navy.mil/how-afm/how-afm.htmlhttp://www.physics.sfasu.edu/afm/afm.htm
LAYERED HARD CRYSTALLINE SOLID MATERIALS
AFM : First high resolution images
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INTERVAL Au COATING :
homogeneous, smoother smaller
polydomain microstructure
Si chip
Si3N4cantilever
TOP VIEW
AFM: Tip Functionalization
ONE-TIME Au COATING :
heterogeneous, rougher larger
polydomain microstructure
1. Gold coatingPurpose:
Methods:
100 nm SIDE VIEW
100 nm SIDE VIEW
TOP VIEW
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microfabricated Si3N4 probe tip
--
--
-
---
--
--
---
-
-+
+
+
+
++
synthetic polymers
polyelectrolytes
self-assembling monolayer
ligands
proteins
• Molecular Elasticity of Individual Polymer Chains• Protein Folding • DNA Interatomic Bonds • Receptor-Ligand Interactions• Covalent Bonds • Colloidal forces • Van der Waals forces • Hydration forces • Hydrophobic forces • Surface Adhesion • Nanoindentation• Electrostatic DLVO forces • Cell Adhesion• Steric Forces of Polymer Brushes
AFM: Tip Functionalization2. Chemical coating
Purpose:
Methods:
Applications:
antibodies
http://www.di.com/AppNotes/LatChem/LatChemMain.html
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(c)
(d)nanotube with
individual ligand
(a) Benoit, M.; Gabriel, D.; Gerisch, G.; Gaub, H. E. Nature Cell. Bio 2000, 2 (6), 313.(b) Ong, Y-L.; Razatos, A.; Georgiou, G.; Sharma, M. K. Langmuir 1999, 15, 2719.
(c) J . Seog, Ortiz/ Grodzinsky Labs 2001(d) Wong S.S.; Joselevich E.; Woolley, A.T.; Cheung, C. L.; Lieber, C. M. Nature 1998, 394 (6688), 52.
(a) Single Cell Dictyostelium Discoideum
(b) E. Coli Bacteria
(c)colloidal particle
AFM: Tip Functionalization
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IV. Chemical Force Microscopy (CFM) Frisbie, et al., 1994
Noncontact (NC) 1995
II. Friction or Lateral Force Microscopy (FFM/ LFM) Frisbie, et al., 1994
I. Normal Force Microscopy
III. Force / Volume Adhesion Microscopy Radmacher, et al., 1994
Contact DC and AC (Force Modulation Microscopy (FMM), Phase Imaging): Hansma, et al., 1991
Intermittant Contact/Tapping / Lift (AC): Hansma, et al., 1994
X=-OH,-CH3, -NH2
XX
XX
XXXXXXXXXXXXX
XXX
Surface Maps:Topography & Roughness, Electrostatic
Interactions, FrictionChemical, Adhesion , Hardness,
Elasticity /ViscoelasticityDynamic Processes :
Erosion, Degradation, Protein-DNA Interactions
AFM: Applications of modes
Timeline:
http://www.di.com/AppNotes/ForceVol/FV.array.html
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PROBE TIP SHARPNESSSheng, et al. J. Microscopy 1999, 196, 1.CANTILEVER
THERMAL NOISELindsay Scanning Tunneling Microscopy
and Spectroscopy 1993, 335.Shao, et al. Ultramicroscopy 1996,
66, 141.
PIEZO AMPLIFIER, SENSOR AND CONTROL ELECTRONICS,
MECHANICAL PARAMETERSPhysik Instruments, Nanopositioning 1998
SPECIMEN DEFORMATION &
THERMAL FLUCTUATIONS
Hoh, et al. Biophys. J. 1998, 75, 1076.
ADHESION FORCEYang, et al. Ultramicroscopy 1993, 50, 157
(*http://cnst.rice.edu/pics.htmlLieber, et al., 2000)
AFM: Resolution factors/Artifact sources
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Biological Applications: AFM Images of CellsContact mode image of human red blood cells - note cytoskeleton is
visible. blood obtained from Johathan Ashmore, Professor of Physiology University College, London. A false color table has been used here, as professorial blood is in fact blue. 15µm scan courtesy M. Miles and J.
Ashmore, University of Bristol, U.K.
Rat Embryo Fibroblast(*M. Stolz,C. Schoenenberger, M.E. Müller Institute,
Biozentrum, Basel Switzerland)
Height image of endothelial cells taking in fluid using Contact Mode AFM. 65 µm scan courtesy J. Struckmeier, S. Hohlbauch, P. Fowler, Digital Intruments/Veeco Metrology, Santa Barbara, USA.
Red Blood CellsShao, et al., : http://www.people.virginia.edu/~js6s/zsfig/random.html
Radmacher, et al., Cardiac Cellshttp://www.physik3.gwdg.de/~radmacher/
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• rest cantilever on top of cell and monitor cantilever deflection up and down = beating of cell
• I. confluent layer of cells : beat regularly in terms of frequency and amplitude, enormous stability of pulsing, cell are synchronized and coupled together : diverse pulse shapes due to macroscopic moving centers of contraction and relaxation• II. individual cell : sequences of high mechanical activity alternate with times of quietness, irregular beating which often last for minutes, active sequences were irregular in frequency andamplitude• III. group of cells: “pulse mapping”
Biological Applications: Manipulation of Living Cells
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http://www.people.virginia.edu/~js6s/zsfig/DNA.html
AFM image of short DNA fragment with RNA polymerase molecule bound to transcription
recognition site. 238nm scan size. Courtesy of Bustamante Lab, Chemistry Department, University
of Oregon, Eugene OR
Image of PtyrTlac supercoiled DNA. 750 nm scan courtesy C. Tolksdorf, Digital
Instruments/Veeco, Santa Barbara, USA, and R. Schneider and G. Muskhelishvili, Istitut für
Genetik und Mikrobiologie, Germany.
TappingMode image of nucleosomal DNA was the highlight of the "Practical Course on Atomic Force Microscopy in
Biology," held at the Biozentrum in Basel, Switzerland, July 1998. Image courtesy of Y. Lyubchenko.
The high resolution of the SPM is able to discern very subtle features such as these two linear dsDNA
molecules overlapping each other. 155nm scan. Image courtesy of W. Blaine Stine
Biological Applications: AFM Images of DNA
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AFM: From Nano to MicroStructures
Human hair (C. Ortiz)
Eggshell
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DIC (Differential Interference Contrast) image of human
lymphocyte metaphase chromosomes on microscopy
slide
dimensions 83 µm * 83 µm
DIC (Differential Interference Contrast) image of human lymphocyte metaphase chromosomes on microscopy slide
dimensions 83 µm * 83 µm
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What does the future hold?
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Atomic Force Microscopy;The Sample Preparation
Pooria GillPhD of Nanobiotechnology
Assistant Professor at [email protected]
In The Name of Allah
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AFM sample preparation
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19
Liquid AFM Images
41 45 48 56 60
70 nm t=0 min 20 2212
Effect of DNase I enzyme on G4-DNA (0.5:1) complex, the complex was immediately adsorbed onto mica and imaged until stable images were obtained, then the DNase I was introduced.
Nucleic Acids Research, 2003, Vol. 31, No. 14 4001-4005
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Humidity affects the adhesion
AFM probeSalbutamol
Measurement of particle-particle interacti
onLactose1µm
Force (nN)
0
200
400
600
800
1000
1200
<10% 22% 44% 65%
‘Nanoscale’ contact‘Macroscale’ contact
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Environmental AFM
Both temperature andhumidity can be controlledin this environmentalchamber.
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